The ability of the gonococcus to gain access to the intracellular environment is important for its ability to initiate and sustain infection. This proposal will focus on two aspects of pathogenesis which appear to be important for the gonococcus. These are the intracellular trafficking in order to acquire the substrate, cytidine monophosphate-N-acetyl neuraminic acid (CMP-NANA) for sialylation of the lipooligosaccharide (LOS) and factors involved in focal actin polymerization seen upon entry of the gonococcus into urethral cells. CMP-NANA is available to the organism within only two intracellular compartments, the Golgi complex and the nucleus. In order for the LOS to become sialylated, the gonococcus must traffic to these locations in the cell. Our studies indicate that the Golgi is the site responsible for providing the CMP-NANA. One mechanism for trafficking involves specific motifs on the cytoplasmic tails of receptors which are involved in clathrin dependent receptor-mediated endocytosis. We have demonstrated the presence of such a receptor, the asialoglycoprotein receptor (ASGP-R), in human urethral epithelial cells and have shown that it is upregulated during gonococcal infection. This receptor binds the terminal galactose on gonococcal LOS. Thus, the first two hypotheses upon which this project is based are that the gonococcus moves in a directed fashion within the human urethral epithelial cell and that this movement is controlled either by the ASGP-R or the LOS through blebbing of the outer membrane. In the second specific aim, we will study a two component system (2CR) which is involved in the control of blebbing. We will perform studies to measure the regulation of this 2CR system in the intracellular environment. Our third hypothesis is that gonococcal porin interacts with actin to facilitate entry of the gonococcus into the eukaryotic cells. We will study the significance of this interaction at the pathogenic and biochemical levels. These three hypotheses will be resolved by the following specific aims: 1- studies will be performed to define the intracellular trafficking pathway of Neisseria gonorrhoeae and the role of ASGP-R and LOS in this trafficking; 2- studies will be undertaken to study the role of the BasS/BasR two component regulator in control of outer membrane blebbing in vivo and in vitro, and 3- biologic and biochemical studies will be performed to elucidate the interactions between Neisserial porin and actin and the role these interactions play in pathogenesis.